Several important features characterize the CD45 leukocyte common antigen: different isoforms which are expressed selectively by the individual leukocyte subpopulations, a cytoplasmic domain with protein tyrosine phosphatase activity that actson substrates involved in cell activation and physical association with lymphocyte antigen receptor complexes. The structure of CD45, which consists of a variably sized extracellular domain due to differential exon splicing, a transmembrane domain and a large cytoplasmic domain with PTPase activity, resembles a receptor capable of signal transduction. Evidence for such a role for CD45 is now emerging in several lymphocyte systems. The focus of this research is on the highly regulated, cell type specific variably spliced forms of the extracellular domain. The overall goal is to determine the functional roles of the different CD45 isoforms with respect to the molecular interactions of CD45 at the cell surface and the intracellular signalling events. The research plan of this application is designed to build on the investigators' findings of selective CD45 exon usage in individual leukocyte subpopulations. The strategy is to use targeted mutations in CD45 exons in the mouse genome to generate mice that are unable to make certain CD45 isoforms. The exons to be functionally deleted are exon 3, an exon common to all isoforms, and exons 4, 5, 6, and 7, the major exons involved in differential splicing to generate the various isoforms. The knock-out of exon 3 can be expected to delete completely CD45 expression.A termination codon introduced into a differentially spliced exon is expected to delete all isoforms containing that exon. The knock-outs will be performed in embryonic stem cells (ES cells) which can be used to generate mutant mice. The possible involvement of CD45 isoforms in developmental parameters, potential disease states, functional capacities of the individual cell types and molecular associations at the cell surface will then be determined.